Dual use detectors for flow cytometry

ABSTRACT

An optical alignment system for aligning a light beam with a core flow in a flow stream. The flow stream may have a sheath fluid and a core flow, where the core flow has a current position within the flow stream. A light source may be used to produce a light beam, and an optical element may be used to direct the light beam at the core flow. In some illustrative embodiments, an actuator is provided for moving the optical element, light source and/or flow stream such that the light directed by the optical element is aligned with the current position of the core flow.

This Application is a continuation of co-pending U.S. patent applicationSer. No. 10/824,859, filed Apr. 14, 2004, and entitled “OPTICALALIGNMENT SYSTEM FOR FLOW CYTOMETRY”; which is a continuation-in-partapplication of co-pending U.S. patent application Ser. No. 10/225,325,filed Aug. 21, 2002; which is a continuation-in-part application of U.S.patent application Ser. No. 09/630,927, filed Aug. 2, 2000, and entitled“OPTICAL DETECTION SYSTEM FOR FLOW CYTOMETRY”, now U.S. Pat. No.6,549,275; which are all incorporated herein by reference.

BACKGROUND

The present invention relates generally to alignment systems, and moreparticularly, to optically aligning a light beam with the core flow of aflow stream.

SUMMARY

The present invention is directed at an optical alignment system foraligning a light beam with a core flow of a flow stream. A flow streammay include a sheath fluid and a core flow, where the core flow has acurrent position within the flow stream. A light source may be used toproduce a light beam, and an optical element may be used to direct thelight beam at the core flow. In some illustrative embodiments, anactuator is provided to move the optical element, light source and/orflow stream such that the light directed by the optical element isaligned with the current position of the core flow.

BRIEF DESCRIPTION OF THE DRAWINGS

Other objects of the present invention and many of the attendantadvantages of the present invention will be readily appreciated as thesame becomes better understood by reference to the following detaileddescription when considered in connection with the accompanyingdrawings, in which like reference numerals designate like partsthroughout the figures thereof and wherein:

FIG. 1 is a schematic diagram showing an illustrative embodiment of thepresent invention;

FIG. 2 is a perspective view of an illustrative portable cytometer inaccordance with the present invention;

FIG. 3 is a schematic view of the illustrative portable cytometer ofFIG. 2;

FIG. 4 is a more detailed schematic diagram showing the portablecytometer of FIG. 3 with the cover not yet depressed;

FIG. 5 is a more detailed schematic diagram showing the portablecytometer of FIG. 3 with the cover depressed;

FIG. 6 is a schematic diagram showing the formation of a flow stream bythe hydrodynamic focusing block 88 of FIG. 4;

FIG. 7 is a schematic diagram showing an illustrative embodiment of thepresent invention;

FIG. 8 is a timing diagram showing an illustrative method for activatingthe light sources of FIG. 7.

FIG. 9 is a schematic diagram showing three separate arrays of lightsources and detectors, each positioned along a different light sourceaxis relative to the central flow axis of the flow stream of FIG. 6;

FIG. 10 is a schematic diagram showing another illustrative embodimentof the present invention which uses a mechanical actuator to align thefirst object relative to the second object;

FIG. 11 is a schematic diagram showing another illustrative embodimentof the present invention which uses a mechanical actuator to align alight source and/or light detector relative to the second object;

FIG. 12 is a schematic diagram showing overlapping elongated beam spotsprovided by an illustrative beam former;

FIG. 13 is a graph showing the light illumination intensity for twospaced laser sources, each producing a beam spot having a Gaussian peaklight intensity;

FIG. 14 is a graph showing the light illumination intensity for twospaced laser sources after the light has been provided through a beamformer in accordance with the present invention;

FIG. 15 is a schematic diagram showing an illustrative beam former foruse with a single light source;

FIG. 16 is a schematic diagram showing an illustrative beam former foruse with a linear array of light sources;

FIG. 17 is a schematic diagram showing a number of illustrativescenarios for detecting the alignment of the cartridge relative to thebase and/or cover;

FIG. 18 is a schematic diagram showing an illustrate method fordetecting the alignment of the core flow in the flow channel and formaking scatter measurements;

FIG. 19 is a schematic diagram of a laminated cartridge having a flowchannel 502 and one or more light blocking layers or regions;

FIG. 20 is a cross-sectional side view of the cartridge of FIG. 19;

FIG. 21 is a schematic diagram of an illustrative object that has alight scattering element provided thereon or therein;

FIG. 22 is a cross-sectional side view of the light scattering elementof FIG. 21;

FIG. 23 is a schematic diagram showing an illustrative embodiment of thepresent invention which uses a mechanical actuator to align a light beamwith the core flow of a flow stream;

FIG. 24 is a schematic diagram showing another illustrative embodimentof the present invention which uses a mechanical actuator to align alight beam with the core flow of a flow stream;

FIG. 25 is a schematic diagram showing yet another illustrativeembodiment of the present invention which uses a mechanical actuator toalign a light beam with the core flow of a flow stream;

FIG. 26 is a schematic diagram showing another illustrative embodimentof the present invention which uses a mechanical actuator to align alight beam with the core flow of a flow stream; and

FIG. 27 is a schematic diagram showing another illustrative embodimentof the present invention which uses a mechanical actuator to align alight beam with the core flow of a flow stream.

DETAILED DESCRIPTION

FIG. 1 is a schematic diagram showing an illustrative embodiment of thepresent invention. The illustrative embodiment includes a first object 2and a second object 3, wherein the second object 3 includes a slot 4 forreceiving the first object 2. While a slot 4 is used in this example, itis not required and some embodiments may not include a slot. The secondobject 3 shown in FIG. 1 includes a linear array of light sources 5 aand a linear array of light detectors 6 a. While a linear array is usedin this example, any suitable array or configuration may be used. Eachlight source is represented by a plus sign (+) and each detector isrepresented by a box. The light sources 5 a may include, for example,Vertical Cavity Surface Emitting Lasers (VCSELs), edge emitting lasers,Light Emitting Diodes (LEDs), an end of an illuminated optical fiber, orany other suitable light source. The light detectors 6 a may include,for example, photo diodes or any other suitable light detector. Thedetectors 6 a may be square, circular, annular or any other suitableshape, as desired. In addition, the detectors 6 a may be a single orsmall number of detectors that detect light from a wide range oflocations. In some cases, optics may be used to direct the light fromthe wide range of locations to the single or small number of detectors,as further described below with respect to FIG. 16.

In the embodiment shown, the linear array of light sources 5 a aremounted on one side (e.g. upper side) of the slot 4 in the second object3, and the linear array of light detectors 6 a are mounted on anopposite side (e.g., lower side) of the slot 4 of the second object 3.However, in some embodiments, the light sources 5 a and the lightdetectors 6 a may be mounted on the same side of the slot 4, such aswhen the light scattering elements are reflective. The pitch and/orspacing of the linear array of light sources 5 a and light detectors 6 amay be set to achieve the desired accuracy of alignment detection, asdesired.

In FIG. 1, the first object 2 includes an elongated light scatteringelement 7 a that extends substantially perpendicular to the linear arrayof light sources 5 a and light detectors 6 a when the first object 2 isinserted into the slot 4 of the second object 3. The term “lightscattering element”, as used herein, may include any optical elementthat diverts, changes, reflects, refracts, absorbs, or otherwise altersa light beam. The one or more light scattering elements 7 a may include,for example, one more lenses, edges or steps, diffraction gratings,absorptive filters, reflectors, flow channels, or any other type oflight scattering element. Other portions of the first object 2 may beclear, opaque or substantially non-transparent, as desired.

In the illustrative embodiment shown in FIG. 1, each of the lightsources 5 a is adapted to provide a light beam that is directed towardthe slot 4 and to one or more corresponding detectors 6 a. The lineararray of light sources 5 a may be positioned with respect to the slot 4so that as long as the first object 2 and second object 3 are alignedwithin a predetermined range 8, one or more of the light beams willintersect at least one of the light scattering elements 7 a, which thenproduces a scattered light profile at one or more of the correspondingdetectors 6 a. The detectors 6 a may be positioned such that at leastone of the detectors 6 a will detect the scattered light profile. Acontroller 9 may be used to identify which of the light sources actuallyproduced the detected scattered light profile, and may correlate thelocation of the identified light source (s) to an alignment position ofthe first object 2 relative to a second object 3.

During operation, and in one illustrative embodiment, each of the lightsources 5 a or a sub-set of light sources may be sequentially activatedby the controller 9. Depending on the alignment of the first object 2relative to the second object 3, a particular light source 5 a or lightsources may produce a light beam that intersects the light scatteringelement 7 a. The light source 5 a or light sources that produce thelight beam that intersects the light scattering element 7 a can beidentified by monitoring the output of the corresponding detectors 6 a.By only activating one or a sub-set of light sources 5 a at any giventime, the light source 5 a or light sources that produced the light beamthat intersects the light scattering element 7 a may be more easilyidentified. However, it is contemplated that all of the light sourcesmay be simultaneously activated and still be within the scope of thepresent invention. In any event, by knowing which light source 5 a orlight sources produced the light beam that intersects the lightscattering element 7 a, and the location thereof, the alignment of thefirst object 2 relative to the second object 3 can be determined.

If the light scattering element 7 a is uniform along its length in theX-direction (e.g. the left-right direction), the linear array of lightsources 5 a and the detectors 6 a may be used to determine the alignmentposition of the first object 2 relative to the second object 3 in the Ydirection (e.g. the up-down direction in FIG. 1). If, however, the lightscattering element 7 a is not uniform along its length, and adapted toproduce a different light scatter profile depending on where the lightstrikes the light scattering element 7 a along its length, the lineararray of light sources 5 a and the detectors 6 a may be used todetermine the alignment position of the first object 2 relative to thesecond object 3 in both the X and Y direction. In this embodiment, thecontroller 9 may not only identify which of the light sources actuallyproduced the detected scattered light profile to determine the Yposition, as described above, but also may correlate the particularlight scatter profile that is detected to an X position.

Alternatively, or in addition, a second elongated light scatteringelement 7 b may be secured relative to the first object 2. The secondelongated light scattering element 7 b may extend in the Y direction,with a second linear array of light sources 5 b and light detectors 6 bextending substantially perpendicular to the second elongated lightscattering element 7 b. Then, the second linear array of light sources 5b and light detectors 6 b may be used in conjunction with the secondelongated light scattering element 7 b to determine the X position ofthe first object 2 relative to the second object 3. In some embodiments,the second elongated light scattering element 7 b may be non-uniformalong its length to help also identify the Y position of the firstobject 2 relative to the second object 3, if desired. If either or bothof the first light scattering element 7 a and the second lightscattering element 7 b are non-uniform along their length, some level orredundancy may be provided in the optical alignment detection system.

It is contemplated that the first object 2 and the second object 3 maybe any type of objects. In one example, the first object 2 may be aremovable media component such as a removable print cartridge, aremovable data storage cartridge such as a removable tape cartridge orremovable flash memory cartridge, a removable bio-analysis cartridge orslide or any other form of removable object. The second object may thenaccept the removable media. Beyond removable media applications, opticalfiber alignment applications, component alignment applications, as wellas many other applications are also within the scope of the presentinvention.

FIG. 2 shows an illustrative embodiment of the present invention thatincludes a removable bio-analysis cartridge. FIG. 2 is a perspectiveview of an illustrative portable cytometer 10, which includes a housing12 and a removable or replaceable cartridge 14. The illustrative housing12 includes a base 16, a cover 18, and a hinge 20 that attaches the base16 to the cover 18. The base 16 includes an array of light sources 22,associated optics and the necessary electronics for operation of thecytometer. The cover 12 includes a manual pressurizing element,pressure-chambers with control microvalves, and an array of lightdetectors 24 with associated optics.

The removable cartridge 14 preferably receives a sample fluid via asample collector port 32. A cap 38 may be used to protect the samplecollector port 32 when the removable cartridge 14 is not in use. Theremovable cartridge 14 preferably performs blood dilution, red celllysing, and hydrodynamic focusing for core formation. The removablecartridge 14 may be constructed similar to the fluidic circuitsavailable from Micronics Technologies, some of which are fabricatedusing a laminated structure with etched channels.

The removable cartridge 14 is inserted into the housing when the cover18 is in the open position. The removable cartridge 14 may include holes26 a and 26 b for receiving registration pins 28 a and 28 b in the base16, which may help provide alignment and coupling between the differentparts of the instrument. In some embodiments, the holes 26 a and 26 band registration pins 28 a and 28 b are not required or even desired,and the alignment detection system described herein is used to detectthe alignment of the removable cartridge 14 with respect to the base 16and cover 18. The removable cartridge 14 may also include a transparentflow stream window 30, which is in alignment with the array of the lightsources 22 and light detectors 24, and one or more light scatteringelements (not shown). When the cover is moved to the closed position,and the system is pressurized, the cover 18 provides controlledpressures to pressure receiving ports 34 a, 34 b, and 34 c in theremovable cartridge 14 via pressure providing ports 36 a, 36 b and 36 c,respectively.

To initiate a test, the cover 18 is lifted and a new cartridge 14 isplaced and registered onto the base 16. A blood sample is introducedinto the sample collector 32. The cover 18 is closed and the system ismanually pressurized. Once pressurized, the instrument performs a whiteblood cell cytometry measurement. The removable cartridge 14 providesblood dilution, red cell lysing, and hydrodynamic focusing for coreformation. The light sources 22, light detectors 24 and associatedcontrol and processing electronics perform solid state alignmentdetection and correction for the particular position of cartridge 14, aswell as differentiation and counting of white blood cells based on lightscattering signals. Rather than using a hinged construction for thehousing 12, it is contemplated that a sliding cartridge slot or anyother suitable construction may be used.

FIG. 3 is a schematic view of the illustrative portable cytometer ofFIG. 2. As above, the base 16 may include an array of light sources 22,associated optics and the necessary control and processing electronics40 for operation of the cytometer. The base 16 may also include abattery 42 for powering the cytometer. The cover 12 is shown having amanual pressurizing element 44, pressure-chambers 46 a, 46 b and 46 cwith control microvalves, and light detectors 24 with associated optics.

The removable cartridge 14 may receive a sample fluid via the samplecollector port 32. When pressurized by the cover 18, the removablecartridge 14 performs blood dilution, red cell lysing, and hydrodynamicfocusing for core formation in a preferred embodiment. Once formed, thecore is provided down a flow stream path 50, which passes the flowstream window 30 of FIG. 2. The array of light sources 22 and associatedoptics in the base provide light through the core stream via the flowstream window 30. The detector(s) and associated optics receivescattered and non-scattered light from the core, also via the flowstream window 30. The controller or processor 40 receives output signalsfrom detector(s), and differentiates and counts selected white bloodcells that are present in the core stream.

It is contemplated that the removable cartridge 14 may include a fluidcontrol block 48 for helping control the velocity of each of the fluids.In the illustrative embodiment, the fluid control block 48 includes flowsensors for sensing the velocity of the various fluids and reports thevelocities to the controller or processor 40. The controller orprocessor 40 may then adjust the microvalves associated withpressure-chambers 46 a, 46 b and 46 c to achieve the desired pressuresand thus desired fluid velocities for proper operation of the cytometer.

Because blood and other biological waste can spread disease, theremovable cartridge 14 preferably has a waste reservoir 52 downstream ofthe flow stream window 30. The waste reservoir 52 receives and storesthe fluid of the flow stream in the removable cartridge 14. When a testis completed, the removable cartridge may be removed and disposed of,preferably in a container compatible with biological waste.

FIG. 4 is a more detailed schematic diagram showing the portablecytometer of FIG. 3 with the cover 18 not yet depressed. FIG. 5 is amore detailed schematic diagram showing the portable cytometer of FIG. 3with the cover depressed. The cover 18 is shown having a manualpressurizing element 44, pressure-chambers 46 a, 46 b and 46 c, andcontrol microvalves generally shown at 60. The array of light sourcesand detectors are not shown in these Figures.

There are three pressure chambers 46 a, 46 b and 46 c, one for eachfluid to be pressurized. In the illustrative embodiment, pressurechamber 46 a provides pressure to a blood sample reservoir 62, pressurechamber 46 b provides pressure to a lyse reservoir 64, and pressurechamber 46 c provides pressure to a sheath reservoir 66. The size andshape of each pressure chamber 46 a, 46 b and 46 c may be tailored toprovide the desired pressure characteristics to the corresponding fluid.

Pressure chamber 46 a includes a first pressure chamber 70 and a secondpressure chamber 72. A first valve 74 is provided between the firstpressure chamber 70 and the second pressure chamber 72 for controllablyreleasing the pressure in the first pressure chamber 70 to a secondpressure chamber 72. A second valve 76, in fluid communication with thesecond pressure chamber 72, controllably vents the pressure in thesecond pressure chamber 72. Each valve is preferably an array ofelectrostatically actuated microvalves that are individually addressableand controllable. Pressure chambers 46 b and 46 c include similar valvesto control the pressures applied to the lyse reservoir 64 and sheathreservoir 66, respectively. Alternatively, each valve may be an array ofelectrostatically actuated microvalves that are pulse modulated with acontrollable duty cycle to achieve a controlled “effective” flow or leakrate.

The removable cartridge 14 has pressure receiving ports 34 a, 34 b, and34 c for receiving the controlled pressures from the cover 18. Thecontrolled pressures are provided to the blood reservoir 62, lysereservoir 64 and sheath reservoir 66, as shown. The lyse reservoir 64and sheath reservoir 66 are preferably filled before the removablecartridge 14 is shipped for use, while the blood reservoir 62 is filledfrom sample collector port 32. A blood sample may be provided to thesample collector port 32, and through capillary action, the blood sampleis sucked into the blood reservoir 62. Once the blood sample is in theblood reservoir 62, the cover 18 may be closed and the system may bepressurized.

A flow sensor is provided in-line with each fluid prior to hydrodynamicfocussing. Each flow sensor 80, 100 and 102 measures the velocity of thecorresponding fluid. The flow sensors are preferably thermal anemometertype flow sensors, and more preferably microbridge type flow sensor. Anoutput signal from each flow sensor 80, 100 and 102 is provided tocontroller or processor 40.

The controller or processor 40 opens the first valve 74 when thevelocity of the blood sample drops below a first predetermined value andopens the second valve 76 when the velocity of the blood sampleincreases above a second predetermined value. Valves 84, 86, 94 and 96operate in a similar manner to control the velocities of the lyse andsheath fluids.

During operation, and to pressurize the system, the manual pressurizingelement 44 is depressed. In the example shown, the manual pressurizingelement 44 includes three plungers, with each plunger received within acorresponding one of the first pressure chambers. The plungers create arelatively high non-precision pressure in the first pressure chambers.Lower, controlled pressures are built in the secondary chambers byopening the first valves 70, 84 and 94, which produce a controllableleak into the secondary chambers. If two much pressure builds up in thesecondary pressure chambers, the corresponding vent valve 76, 86 and 96are opened to relieve the pressure.

When closing the cover 18, the normally open first valves 74, 84 and 94are closed while the vent valves 76, 86 and 96 are open. When apredetermined pressure P is achieved in the first pressure chambers, thevent valves 76, 86 and 96 are closed, and the first valves 74, 84 and 94are opened to build a lower pressure P′ in the secondary pressurechambers. The controlled pressure in the secondary pressure chambersprovide the necessary pressures to the fluidic circuit of the removablecartridge 14 to produce fluid flow for the blood, lyse and sheath. Thevelocity of the fluid flow is then measured by the downstream flowsensors 80, 100 and 102. Each flow sensor provides an output signal thatis used by the controller or processor 40 to control the operation ofthe corresponding first valve and vent valve to provide a desired andconstant flow rate for each fluid.

Downstream valves generally shown at 110 may also be provided.Controller or processor 40 may close downstream valves 110 until thesystem is pressurized. This may help prevent the blood, lyse and sheathfrom flowing into the fluid circuit before the circuit is pressurized.In another embodiment, downstream valves 110 are opened by mechanicalaction when the cover is closed.

FIG. 6 is a schematic diagram showing the formation of a flow stream andcore by the hydrodynamic focusing block 88 of FIG. 4. The hydrodynamicfocusing block 88 receives blood, lyse and sheath at controlledvelocities from the fluid driver. The blood is mixed with lyse, causingthe red blood cells to be removed. This is often referred to as red celllysing. The remaining white blood cells are provided down a centrallumen 150, which is surrounded by sheath fluid to produce a flow stream50. The flow stream 50 includes a core stream 160 surrounded by thesheath fluid 152. The dimensions of the channel are reduced as shown sothat the white blood cells 154 and 156 are in single file. The velocityof the sheath fluid is preferably about 9 times that of the core stream160. However, the velocity of the sheath fluid and core stream 160preferably remains sufficiently low to maintain laminar flow in the flowchannel.

Light emitters 22 and associated optics are preferably provided adjacentone side of the flow stream 50. One or more light detector(s) 24 andassociated optics are provided on another side of the flow stream 50 forreceiving the light from the light emitters 22 via the flow stream 50.The output signals from the light detector(s) 24 are provided tocontroller or processor 40, wherein they are analyzed to identify and/orcount selected white blood cells in the core stream 160.

FIG. 7 is a schematic diagram showing an array of light sources and anarray of light detectors for analysis of the core stream 160 of FIG. 6,and for identifying the relative alignment position of the cartridge 14relative to the base 16 and/or cover 18 (see, for example, FIG. 2). Thelight sources are shown as plus (+) signs and the detectors are shown asboxes. In the embodiment shown, the array of light sources is providedadjacent one side of the flow stream 50, such as in or on the base 16,and the array of light detectors is provided adjacent the opposite sideof the flow stream, such as in or on the cover 18. Each of the lightdetectors preferably corresponds to one of the light sources. In someembodiments, only a single or small number of light detectors areprovided that are capable of detecting light from a relatively largearea, such as the area corresponding to the array of light sources. Inthe embodiment shown, the array of light sources and the array of lightdetectors are arranged along a light source axis 200 that issubstantially orthogonal to the axis of the flow stream 50. It iscontemplated, however, that the array of light sources and the array oflight detectors may be arranged along a light source axis that is offsetat any angle relative to the axis of the flow stream 50. Although thearray of light sources and the array of light detectors are shown aslinear arrays, any suitable arrangement may be used.

The array of light sources is preferably an array of lasers such asVertical Cavity Surface Emitting Lasers (VCSEL) fabricated on a commonsubstrate. Because of their vertical emission, VCSELs are ideally suitedfor packaging in compact instruments such as a portable cytometer.Preferably, the VCSELs are “red” VCSELs that operate at wavelengths thatare less than the conventional 850 nm, and more preferably in the 670 nmto 780 nm range, but this is not required. Red VCSELs may have awavelength, power and polarization characteristic that is ideally suitedfor scatter measurements. It is contemplated, however, that LightEmitting Diodes (LEDs) or any other suitable light source may be used.The light detectors may be, for example, photo diodes or any othersuitable light detector. The detectors may be square, circular, annularor any other suitable shape, as desired.

In some embodiments, each of the light sources is adapted to provide alight beam. To identify the relative alignment position of, for example,the cartridge 14 relative to the base 16 and/or cover 18 (e.g., see FIG.2), the array of light sources may extend a sufficient range so that oneor more of the light beams will intersect at least one of the lightscattering element of the cartridge 14. In the illustrative embodiment,the cartridge 14 includes a number of light scattering elementsincluding, for example, cartridge edge 210, flow channel edge 212, andembossed light scattering elements 214. Each of the light scatteringelements may produce a scattered light profile.

The detectors may be located such that at least one of the detectorswill detect the scattered light profile of at least one of the lightscattering elements. A controller may be used to identify which of thelight sources actually produced the detected scattered light profile,and to correlate the location of the identified light source(s) to analignment position of the cartridge 14 relative to the base 16 and/orcover 18.

During operation, and in one illustrative embodiment, each of the lightsources or a sub-set of light sources may be sequentially activated.Depending on the alignment of the cartridge 14 to the base 16 and/orcover 18, a particular light source or light sources may produce a lightbeam that intersects a light scattering element, such as lightscattering element 214. The light source or light sources that producethe light beam that intersects the light scattering element 214 can beidentified by monitoring the output of the corresponding detectors. Byonly activating one or a sub-set of light sources at any given time, thelight source or light sources that produced the light beam thatintersects the light scattering element 214 may be more easilyidentified. By knowing which light source or light sources produced thelight beam that intersects the light scattering element 214, and thelocation thereof, the alignment of the cartridge 14 relative to the base16 and/or cover 18 can be determined.

FIG. 8 is a timing diagram showing an illustrative method for activatingthe light sources of FIG. 7. In the illustrative embodiment, each of thelight sources is sequentially activated, beginning with the light source220 which is located at the bottom of the array of light sources shownin FIG. 7. The sequential activation of the light sources is showngenerally at 218, where the notation V1, V2, etc., corresponds to theactivation of VCSEL1 220 a, VCSEL2 220 b, etc., of FIG. 7. The responseof the corresponding detectors is shown generally at 224.

When light source 220 a is activated, no scattered light profile isdetected at the corresponding detectors because, as shown in FIG. 7, thecartridge 14 is not situated between light source 220 a and thecorresponding detectors. While FIG. 7 shows three light detectors foreach light source, only the left and right detectors may be used fordetecting a scattered light profile in some embodiments. Light source220 b may then be activated. When this occurs, the correspondingdetectors detect a scatter light profile 222. The characteristics of thescatter light profile 222 may identify the light scattering element asthe cartridge edge 210.

When the third and forth light sources are activated, no scattered lightprofile is detected at the corresponding detectors. When the fifth lightsource 220 c is activated, the corresponding detectors detect a scatterlight profile 224. The characteristics of the scatter light profile 224may identify the light scattering element as an embossed lightscattering element 214. Continuing with the example, when light source220N is activated, the corresponding detectors detect a scatter lightprofile 226. The characteristics of the scatter light profile 226 mayidentify the light scattering element as a fluid channel edge 212. Forillustration purposes, the light scatter profiles 222, 224 and 226 areshown as having differing amplitudes. However, it is contemplated thatany suitable parameter or characteristic may be used to differentiatebetween the light scatter profiles, as desired. Alternatively, only thelocations of the light scattering elements are identified, and nodifferentiation between light scattering elements is provided. In someembodiments, only the light scatter profile 224 of the embossed lightscattering element 214 may be identified, and the detection of the otherlight scattering elements may be disregarded.

Once this relative alignment of the cartridge 14 is determined, thepresent invention may identify which of the one or more light sourceand/or light detector elements have a location that is adjacent the flowstream 50. For example, in the illustrative embodiment of FIG. 7, thepresent invention may identify light sources 220×, 220 y and 220 z ashaving a location that is adjacent the flow stream 50. Depending on therelative alignment of the cartridge 14 and the base 16 and/or cover 18,different light sources and/or light detectors may be selected. Forexample, if the cartridge 14 were moved up so that light source 220 bwere positioned above the embossed light scattering element 214, thenthe three light sources immediately above light source 220 c would havea location adjacent the flow stream 50, and would be selected. Once thelight sources have been identified and selected, the selected lightsources and/or light detectors may be used to, for example, detect oneor more parameters and/or characteristics of the flow stream.

FIG. 9 shows another illustrative embodiment of the present invention.This embodiment includes three separate arrays of light sources andlight detectors. While three arrays are shown, it is recognized that anysuitable number may be used, depending on the application. In theillustrative embodiment, each array of light sources and light detectorsis positioned along a different light source axis relative to thecentral flow axis of the flow stream.

A first array of light sources and light detectors is shown at 300. Inthe illustrative embodiment shown, the light sources and light detectorsof the first array 300 are arranged in a linear array along a firstlight source axis. The array of light detectors is positioned in linewith the linear array of light sources. The light sources and lightdetectors of the first array 300 may be used to measure, for example,the lateral alignment of the cells in the flow stream 50, the particlesize, and in some cases, the velocity of the particles. Alternatively,or in addition, the first array of light sources and light detectors 300may be used to detect the position of a light scattering element, suchas light scattering element 312, to help determine the alignment of thecartridge 14 relative to the base 16 and/or cover 18. For example, thelight scattering element 312 may produce a light scattering profile thatcan be detected by one or more corresponding detectors. Once thelocation of the light scattering element 312 is identified, thealignment of the cartridge 14 relative to the base 16 and/or cover 18can be determined.

A second array of light sources and light detectors is shown at 302. Thesecond array of light sources may be arranged in a linear array along asecond light source axis relative to the flow axis of the flow stream50. In the illustrative embodiment, the light detectors of the secondarray 302 include three linear arrays of light detectors. One lineararray of light detectors is positioned in line with the linear array oflight sources. The other two linear arrays of light detectors are placedon either side of the in-line array of light detectors. The second arrayof light sources and light detectors 302 is similar that shown anddescribed with respect to FIG. 7. As detailed with respect to FIG. 7,the second array of light sources and light detectors 302 may be usedto, for example, help determine the relative alignment of the cartridge14 with the base 16 and/or cover 18.

Once the relative alignment of the cartridge 14 is determined, one ormore light source and/or light detector elements located adjacent theflow stream 50 may be identified. Once these light sources have beenidentified and selected, the selected light sources and correspondinglight detectors may be used to, for example, detect one or moreparameters and/or characteristics of the flow stream. In oneillustrative embodiment, the selected light sources and light detectorsof the second array 302 may be used to measure the small anglescattering (SALS) produced by selected particles in the flow stream 50.In this case, the outer light detectors may be spaced sufficiently fromthe in-line detector to intercept the small angle scattering (SALS)produced by selected particles in the flow stream 50.

It is contemplated that the in-line detectors of the second array oflight sources and light detectors 302 may be used to detect the lightthat is not significantly scattered by the particles in the core stream.Thus, the in-line linear array of light detectors of the second array302 may be used to provide the same measurements as the in-line array ofdetectors of the first array 300, if desired. The measurements of bothin-line arrays of detectors may be compared or combined to provide amore accurate result. Alternatively, or in addition, the in-linedetectors of the second array 302 may be used as a redundant set ofdetectors to improve the reliability of the measurement.

The in-line detectors of the second array 302 may also be used inconjunction with the in-line detectors of the first array 300 to moreaccurately determine the time-of-flight or velocity of the particles inthe flow stream. The measurement may be more accurate because thedistance between detectors may be greater. As indicated above, byknowing the velocity of the particles, small variations in the flow ratecaused by the fluid driver can be minimized or removed by thecontroller.

A third array of light sources and light detectors 350 is also shown.The third array of light sources and light detectors 350 may be used to,for example, measure the forward angle scattering (FALS) produced byselected particles in the flow stream. In the illustrative embodiment,the light sources are arranged in a linear array along a third lightsource axis relative to the flow axis of the flow stream 50. Each lightsource preferably has a corresponding light detector, and each lightdetector is preferably annular shaped with a non-sensitive region or aseparate in-line detector positioned in the middle. The annular shapedlight detectors may be sized to intercept and detect the forward anglescattering (FALS) produced by selected particles in the flow stream.

If a separate in-line detector is provided, it can be used to providethe same measurement as the in-line detectors of the first array 300and/or second array 302. When so provided, the measurements from allthree in-line arrays of detectors of first array 300, second array 302and third array 350 may be compared or combined to provide an even moreaccurate result. The in-line detectors of the third array 302 may alsobe used as another level or redundancy to improve the reliability of thecytometer.

The in-line detectors of the third array 350 may also be used inconjunction with the in-line detectors if the first array 300 and/orsecond array 302 to more accurately determine the time-of-flight orvelocity of the particles in the flow stream. The measurement may bemore accurate because the distance between detectors may be greater. Asindicated above, by knowing the velocity of the particles, smallvariations in the flow rate caused by the fluid driver can be minimizedor removed by the controller.

By using three separate arrays of light sources and detectors, and insome embodiments, the optics associated with each array may be optimizedfor the desired application. For example, and in some embodiments, theoptics associated with the first array 300 may be designed to providewell-focused laser light on the plane of the core flow. This may helpprovide resolution to the alignment, size and particle velocitymeasurements performed by the first array 300. Likewise, the opticsassociated with the second array 302 may be designed to providewell-focused laser light on the plane of the core flow. Well focusedlight is often desirable when measuring the small angle scattering(SALS) produced by selected particles in the flow stream. Finally, theoptics associated with the third array 350 may be designed to providecollimated light to the core flow. Collimated light may be desirablewhen measuring forward angle scattering (FALS) produced by selectedparticles in the flow stream.

Using arrays of lasers offers a number of important advantages over asingle light source configuration. For example, a linear array of lasersmay be used to determining the lateral alignment of the path of theparticles in the core steam 160. One source of uncertainty in thealignment of the particle stream is the width of the core stream, whichleads to statistical fluctuations in the particle path position. Thesefluctuations can be determined from analysis of the detector data andcan be used by the controller or processor 40 to adjust the valves ofthe fluid driver in order to change the relative pressures that areapplied to the sample fluid and the supporting fluids to change thealignment of the selected particles in the flow stream.

To determine the lateral alignment of the cells in the fluid stream 50,the cells may pass through several focused spots produced by the arrayof light sources (e.g. VCSELs). The cells produce a drop in signal inthe corresponding in-line reference detectors. The relative strengths ofthe signals may be used by the controller or processor 40 to determinethe center of the particle path and a measure of the particle width.

Another advantage of using an array of light sources rather than asingle laser configuration is that the velocity of each cell may bedetermined. Particle velocity can be an important parameter inestimating the particle size from light scatter signals. In conventionalcytometry, the particle velocity is extrapolated from the pump flowrates. A limitation of this approach is that the pumps must be veryprecise, the tolerance of the cytometer flow chambers must be tightlycontrolled, no fluid failures such as leaks can occur, and noobstructions such as micro bubbles can be introduced to disturb the flowor core formation.

To determine the velocity of each cell, the system may measure the timerequired for each cell to pass between two successive spots. Forexample, and with reference to FIG. 9, a cell may pass a detector 208and then detector 210. By measuring the time required for the cell totravel from detector 208 to detector 210, and by knowing the distancefrom detector 208 to detector 210, the controller or processor 40 cancalculate the velocity of the cell. This would be an approximatevelocity measurement. This is often referred to as a time-of-flightmeasurement. Once the velocity is known, the time of travel through thespot on which the particle is approximately centered (a fewmicroseconds) may provide a measure of particle length and size.

It is contemplated that the particle velocity can also be used to helpcontrol the fluid driver. To reduce the size, cost and complexity of acytometer, the replaceable cartridge 14 of FIG. 2 may be manufacturedfrom a plastic laminate or molded parts. While such manufacturingtechniques may provide inexpensive parts, they are typically lessdimensionally precise and repeatable, with asymmetrical dimensions andwider tolerance cross-sections. These wider tolerances may producevariations in particle velocity, particularly from cartridge tocartridge. To help compensate for these wider tolerances, thetime-of-flight measurement discussed above can be used by the controlleror processor 40 to adjust the controlled pressures applied to the blood,lyse and sheath fluid streams such that the particles in the core streamhave a relatively constant velocity. Also, and because of these widertolerances, it is often desirable to determine the alignment of thecartridge 14 relative to the relative to the base 16 and/or cover 18.Once the alignment position is determined, the appropriate light sourcesand light detectors can be selected for analyzing the selectedparameters or characteristics of the flow stream.

To further evaluate the cell size, it is contemplated that laser beamsmay be focused both along the cell path and across the cell path.Additionally, multiple samples across the cell may be analyzed fortexture features, to correlate morphological features to other celltypes. This may provide multiple parameters about cell size that mayhelp separate cell types from one another.

Yet another advantage of using an array of lasers rather than a singlelaser source configuration is that a relatively constant lightillumination may be provided across the flow channel. This may beaccomplished by overlapping Gaussian beams provided by adjacent VCSELs,as shown in FIG. 12. In single laser systems, the light illuminationacross the flow channel typically varies across the channel. Thus, if aparticle is not in the center of the flow channel, the accuracy ofsubsequent measurements may be diminished.

FIG. 10 is a schematic diagram showing another illustrative embodimentof the present invention which uses a mechanical actuator to align thefirst object relative to the second object. The illustrative embodimentincludes a first object 352 and a second object 353, wherein the secondobject 352 includes a slot 354 for receiving the first object 352. Whilea slot 354 is used in this example, it is not required and someembodiments may not include a slot. The second object 353 shown in FIG.1 includes one or more light sources, such as light source 355 and oneor more light detectors, such as light detector 356.

In the embodiment shown, the light source 355 is mounted on one side(e.g. upper side) of the slot 354 in the second object 353, and thelight detector 356 is mounted on an opposite side (e.g., lower side) ofthe slot 354 of the second object 353. Like above, the first object 352may include an elongated light scattering element 357, as shown.

A controller 359 may be used to control a mechanical actuator 361 that,when activated, may move the first object 352 relative to the secondobject 353. In the embodiment shown, the mechanical actuator 361 movesthe first object 352 in an up and/or down direction relative to thesecond object 353. The actuator 361 may be any type of actuatorincluding, for example, a step motor, a micro actuator such as anelectro-statically actuated micro-actuator, or any other suitableactuator, as desired.

During use, the controller 359 may instruct the actuator 361 to move thefirst object 352 relative to the second object 353 until the lightsource 355 produces a light beam that intersects the light scatteringelement 357, which then produces a light scatter profile that can bedetected by light detector 356. Once this occurs, the first object 352may be considered properly aligned with the second object 353. In theillustrative embodiment, the original position of the first object 352is shown by dotted lines, which is moved in a downward direction untilthe light scattering element 357 of the first object 352 is aligned withthe light source 355. In some embodiments, the light scattering element357 may be, for example, one more lenses, edges or steps, diffractiongratings, absorptive filters, reflectors, flow channels, or any othertype of light scattering element.

Rather than moving the first object 352 relative to the second object353, it is contemplated that the light source 355 itself may be movedrelative the second object 353. This is illustrated in FIG. 11. In FIG.11, an actuator 363 moves the light source 355 relative to the secondobject 353, which by definition, also moves the light source 355relative to the first object 352. In the embodiment shown, thecontroller 359 instructs the actuator 363 to move the light source 355until the light source 355 produces a light beam that intersects thelight scattering element 357 on the first object 352, which thenproduces a light scatter profile that can be detected by light detector356. In the illustrative embodiment, the original position of the lightsource 355 is shown by dotted lines at 370, which after actuation, ismoved in a downward direction until the light source 355 is aligned withthe scattering element 357 of the first object 352. In some embodiments,a stationary array of light detectors may be used to detect light acrossa range of locations. In other embodiments, one or more largerstationary detectors may be used to detect light across a range oflocations. In still other embodiments, one or more movable lightdetectors may be used, and moved by the actuator 363 in conjunction withthe light source 355, as shown in FIG. 11.

Referring now to FIG. 12, in some embodiments, the light beams from allor selected light sources may pass through a beam former or the like.When the light sources are in an array that extends along an array axis,the beam former may, for example, increase the beam spot size of eachlight source in the direction of the axis, and in some cases decreasethe beam spot size in a direction perpendicular to the axis. In someembodiments, the beam former may increases the beam spot size in thedirection of the axis such that the light output of each light source atleast partially overlaps the light output of an adjacent light source.For example, FIG. 12 shows a number of beam spots 400 a-400 f that havebeen formed by a beam former, wherein each of the beam spots has beenincreased in the direction of the light source array axis, and decreasedin the direction perpendicular to the light source array axis. Inaddition, each of the beam spots 400 a-400 f at least partially overlapsthe beam spot of an adjacent light source. This increases the distancethat the beam spots 400 a-400 f can collectively span, and increases theuniformity of light illumination across the illuminated area.

FIG. 13 shows the light illumination intensity for two spaced lasersources. Each light source produces a beam spot having a Gaussian peaklight intensity. A dip in light intensity is shown between the lightsources. FIG. 14 shows the light illumination intensity for two spacedlaser sources after the light has been provided through a beam former asdescribed above. Each of the beam spots has been increased in thedirection of the light source array axis, and decreased in the directionperpendicular to the light source array axis. Also, each of the beamspots at least partially overlaps the beam spot of the adjacent lightsource. As can be seen, this may increase the uniformity of lightillumination across the illuminated area.

FIG. 15 shows an illustrative beam former that may be used for one ormore light sources. The light sources are shown at 410, and may providea beam spot to a beam former generally shown at 412. The light sourcesmay be, for example, VCSELs, edge emitting photo diodes, or any othersuitable light source. The beam former 412 includes a first lens 414 anda second lens 416 that may collectively decrease the beam spot size inthe vertical direction, and a third lens 418 that increases the beamspot size in the horizontal direction. The first lens 414, second lens416 and the third lens 418 may collectively focus the elongated beamspot 420 on the plane of the core flow 160 of the flow channel 50 in thecartridge 14, as shown. As can be seen, the beam former 412 may increasethe distance that the beam spots 420 can span, and may increase theuniformity of light illumination across the flow channel 50. Once thelight passes through the core flow 160, the light may be received byanother lens (not shown) such as a diffractive optical element (DOE),and may be directed to one or more detectors for detection and analysis.

FIG. 16 shows an illustrative beam former for use with a linear array oflight sources. The linear array of light sources is generally shown at450, and may include a linear array of VCSELs having an array axis thatextends in a horizontal direction (X-direction) as shown. A flow channelis shown at 50. The flow channel extends in a vertical direction(Y-direction). One or more detectors is shown at 452. Each of the VCSELsin the array of VCSELs 450 preferably provides a beam spot to beamformer 456. The beam former 456 may include a number of lenses or otheroptical elements that collectively form overlapping elongated beamspots, such as those shown in FIG. 12. The illustrative beam former 456may include a first lens 460, a second lens 462 and a third lens 464that collectively decrease the beam spot size in the vertical direction(Y-direction), and a fourth lens 466 that increases the beam spot sizein the horizontal direction. The fourth lens 466 may be, for example, acylinder lens that is concave in the vertical direction (Y-direction).The first lens 460, second lens 462, third lens 464, and the fourth lens466 may collectively focus the overlapping elongated beam spots on theplane of the flow channel 50 in the cartridge 14. As detailed withrespect to FIG. 12, the beam former 456 may increase the distance thatthe beam spots provided by the array of light sources 450 cancollectively span across the cartridge 14, and may increases theuniformity of light illumination across the illuminated area. Once thelight passes through the core flow 160, the light may be collected byanother lens 470, such as a diffractive optical element (DOE), and maybe directed to one or more detector(s) 452 for detection and analysis.

FIG. 17 is a schematic diagram showing a number of illustrativescenarios for detecting the alignment of the cartridge 14 relative tothe base 16 and/or cover 18. To identify the relative alignment positionof the cartridge 14 relative to the base 16 and/or cover 18, the arrayof light sources preferably extend over a sufficient range so that atleast one of the elongated beam spots shown in FIG. 12 intersects atleast one of the light scattering element of the cartridge 14. In theillustrative embodiment shown in FIG. 17, the cartridge 14 includes anumber of light scattering elements including a cartridge edge 210 andtwo flow channel edges 212 a and 212 b. Each of the light scatteringelements preferably produces a scattered light profile.

One or more detectors may be located such that at least one of thedetectors will detect the scattered light profile of at least one of thelight scattering elements. A controller may be used to identify which ofthe light sources actually produced the detected scattered lightprofile, and to correlate the location of the identified light source(s) to an alignment position of the cartridge 14 relative to the base 16and/or cover 18.

In a first scenario, the elongated beam spot region produced by the beamformer is collectively shown at 470. In one example, the collectiveelongated beam spot region 470 is formed by a linear array of ten (10)VCSELs having a 25 micron pitch. The beam former elongates and overlapsthe individual beam spots of the 10 VCSEL devices, and produces thecollective elongated beam spot region 470 with a length of about 720microns at the cartridge 14.

In the first scenario, the cartridge 14 is aligned such that thecollective elongated beam spot region 470 only overlaps one lightscattering element, namely, the cartridge edge 210. If the flow channel50 were within the range of the 720 micron collective elongated beamspot region 470, the location of the cartridge edge 210 could be used toidentify individual VCSELs that are located adjacent the flow channel50. However, in the embodiment shown, the flow channel 50 is not withinthe range of the 720 micron collective elongated beam spot region 470.As such, the processor or controller may indicate that the cartridge 14is misaligned too much to perform an analysis of the flow channel 50.The range covered by the collective elongated beam spot region 470 couldbe extended by simply adding additional light sources, light detectorsand associated optics.

In a second scenario, the cartridge 14 is aligned such that thecollective elongated beam spot region 472 overlaps two light scatteringelements, namely, the cartridge edge 210 and the flow channel edge 212a. Again, if the entire flow channel 50 were within the range of the 720micron collective elongated beam spot region 472, the location of thecartridge edge 210 and/or the flow channel edge 212 a could be used toidentify individual VCSELs that are located adjacent the flow channel50. However, in the embodiment shown, the flow channel 50 is notentirely within the range of the 720 micron collective elongated beamspot region 472. As such, the processor or controller may indicate thatthe cartridge 14 is misaligned too much to perform an analysis of theflow channel 50. The range covered by the collective elongated beam spotregion 472 could be extended by simply adding additional light sourcesand associated optics.

In a third scenario, the cartridge 14 is aligned such that thecollective elongated beam spot region 474 overlaps only one lightscattering element, namely, the flow channel edge 212 a. Again, if theentire flow channel 50 were within the range of the 720 microncollective elongated beam spot region 474, the location of the flowchannel edge 212 a could be used to identify individual VCSELs that arelocated adjacent the flow channel 50. However, in the embodiment shown,the flow channel 50 is not entirely within the range of the 720 microncollective elongated beam spot region 474. As such, the processor orcontroller may indicate that the cartridge 14 is misaligned too much toperform an analysis of the flow channel 50. The range covered by thecollective elongated beam spot region 474 could be extended by simplyadding additional light sources and associated optics.

In a fourth scenario, the cartridge 14 is aligned such that thecollective elongated beam spot region 476 overlaps two light scatteringelement, namely, the flow channel edge 212 a and the flow channel edge212 b. In this scenario, the entire flow channel 50 is within the rangeof the 720 micron collective elongated beam spot region 476. As such,the location of the flow channel edge 212 a and the flow channel edge212 b may be used to identify individual VCSELs that are locatedadjacent the flow channel 50. Once identified, the identified individualVCSELs may be used to determine selected parameters or characteristicsof the flow stream 50.

FIG. 18 is a schematic diagram showing an illustrate method fordetecting the alignment of the core flow in the flow channel 50 and formaking scatter measurements. In the illustrative embodiment, once theVCSELs are identified that are located adjacent the flow channel 50,each of these VCSELs may be sequentially activated to identify thelocation of the core in the flow channel 50 and/or to perform scatteringmeasurements, as shown at 480 a, 480 b and 480 c. Alternatively, or inaddition, all of the identified VCSELs may be simultaneously activatedas shown at 482, and the output of the corresponding detectors may bemonitored to determine the location of the core in the flow channeland/or to perform scattering measurements.

FIG. 19 is a schematic view of a laminated cartridge 500 having a flowchannel 502. FIG. 20 is a cross-sectional side view of the cartridge 500of FIG. 19. The cartridge 500 includes a number of laminations,including a bottom lamination 504, a top lamination 506 and one or moreintermediate laminations 508. The flow channel 502 may be formed by anetched channel in one or more of the intermediate laminations 508. Tohelp detect a cartridge edge 510, a channel edge 512, or some otherfeature, one or more light blocking layers or regions may be included inor on one of laminated layers. For example, a light blocking layer orregion 514 may be provided on top of the top lamination 506 as shown.The light blocking layer or region 514 may be, for example, a sticker orother filter that is attached to the top and/or bottom surface of thecartridge 500. Alternatively, the light blocking layer may beincorporated into one of the intermediate laminations, as shown at 509,if desired.

The light blocking layer or region may extend, for example, between thecartridge edge 510 and the channel edge 512. The light blocking layer orregion 514 may prevent light that is emitted by a light sourcepositioned between the cartridge edge 510 and the channel edge 512 fromreaching the corresponding detector(s). This may simplify the detectionof the cartridge edge 510 and/or the channel edge 512, because detailedscattering profiles may not need to be analyzed. Instead, a simplerlight/no-light algorithm may be used. It is recognized that the lightblocking layer or region need not extend between the cartridge edge 510and the channel edge 512. Rather, it is contemplated that anyarrangement suitable for detecting the relative position of thecartridge 500 may be used.

FIG. 21 is a schematic diagram of an illustrative object 600 that has alight scattering element 602. FIG. 22 is a cross-sectional side view ofthe light scattering element 602 of FIG. 21. A light source 604 (shownas a “+” sign in FIG. 21) is shown positioned above the light scatteringelement 602, and an array of detectors 606 (shown as boxes in FIG. 21)are shown positioned below the light scattering element 602. The lightsource 604 preferably directs a light beam toward the light scatteringelement 602, and depending the relatively alignment of the lightscattering element 602 to the light source 604, the light scatteringelement 602 may direct the light beam to one or more of the detectors606. In one example, and referring to FIG. 22, if the light source ispositioned at position 604 a relative to the light scattering element602, the light scattering element 602 may direct the light beam todetector 606 a. If the light source is positioned at position 604 brelative to the light scattering element 602, the light scatteringelement 602 may direct the light beam to a detector 606 b. If the lightsource is positioned at position 604 c relative to the light scatteringelement 602, the light scattering element 602 may direct the light beamto a detector 606 c. As such, by monitoring which of the detectors 606detects the light beam, the relative position of the light source 604and the light scattering element 602 and thus the object 600 can bedetermined. In one embodiment, the light scattering element 602 is alens. However, any suitable light scattering element may be used. It iscontemplated that the light scattering element 602 may be used todetermine the relative alignment of the object 600 in either one- ortwo-dimensions.

FIG. 23 is a schematic diagram showing an illustrative embodiment of thepresent invention which uses a mechanical actuator to align a light beamwith the core flow of a flow stream. This illustrative embodimentincludes a light source 700 for producing a light beam 702, an opticalelement 704 for focusing the light beam 702 on the core flow 706 of aflow stream, and a detector 708 for detecting scattered and/or reflectedlight 710 from the core flow 706. The optical element 704 is shownschematically as a lens, but it may include a set of lenses or any othersuitable optical element, as desired. It is also contemplated thatanother optical element (not shown in FIG. 23) may be provided betweenthe core flow 706 and the detector 708 in some cases, as shown in forexample FIG. 25-27. Also, it is contemplated that the detector 708 maybe position on the same side as the light source, if desired.

The core flow 706 is included in a flow stream traveling down a flowchannel 712. The flow channel 712 shown in FIG. 23 is flowing into thepage. The core flow 706 may include a sheath fluid (liquid or gas)flowing on either side of the core flow 706. In some embodiments, thesheath fluid and core flow 706 have laminar flow as they pass throughthe flow channel 712.

As shown generally at 720, the core flow 706 may be relatively centeredin the flow channel 712. However, under some conditions, the core flow706 may not flow down the center or at some other predetermined positionin the flow channel 712. For example, as shown generally at 722, thecore flow 706 may flow left of center of the flow channel 712. Likewise,as shown generally at 724, the core flow 706 may flow right of center ofthe flow channel 712.

To help compensate for the various possible positions of the core flow706 within the flow channel 712, it is contemplated that an actuator 726or the like may be used to move the optical element 704 so that thelight beam 702 emitted by the light source 700 is aligned with (e.g.focused on) the current position of the core flow 706 in the flowchannel 712. The actuator 726 may be controlled by a controller 728. Insome cases, the controller 728 may receive one or more feedback signalsindicating whether the light beam 702 is currently aligned with (e.g.focused on) the current position of the core flow 706 in the flowchannel 712. If not, the controller 728 may instruct the actuator tomove the optical element 704 until the light beam 702 is aligned with(e.g. focused on) the current position of the core flow 706 in the flowchannel 712. The feedback signal may include, for example, an outputsignal from the detector 708.

In one example, and as generally shown at 722, when the core flow 706 isleft of center of the flow channel 712, the controller 728 may instructthe actuator 700 to move the optical element 704 to the left, which maydirect the light beam 702 at the current position of the core flow 706in the flow channel 712. Likewise, and as generally shown at 724, whenthe core flow 706 is right of center of the flow channel 712, thecontroller 728 may instruct the actuator 700 to move the optical element704 to the right, which may direct the light beam 702 at the currentposition of the core flow 706 in the flow channel 712. In some cases,the controller 728 may instruct the actuator 700 to first move theoptical element 704 to identify an edge of the flow channel 712. Thismay be considered a coarse alignment. In some cases, the flow channel712 is part of a fluidic cartridge, and the fluidic cartridge isnon-transparent except at the flow channel. Thus, as the light beam 702is directed across an edge of the flow channel 712, an abrupt change inlight intensity at the detector may occur. Then, the controller 728 mayinstruct the actuator 700 to move the optical element 704 to direct thelight beam 702 at the current position of the core flow 706 in the flowchannel 712.

The actuator 726 may be any type of mechanical actuator. In some cases,the actuator 726 may be a stepper motor, a voice coil, an electrostaticactuator, a magnetic actuator, a micro-positioning actuator similar tothat shown and described in U.S. Pat. No. 6,445,514, or any othersuitable actuator, as desired.

In some embodiments, the light source 700 may include a single lightsource. In other embodiments, the light source may include more than onelight source, such as an array of light sources. In some cases, and whenthe light source shown at 700 includes more than one light source, atleast some of the light sources may produce different wavelengths oflight, if desired. The different wavelengths of light may be emitted andimaged onto the core flow by the optical element, as discussed above.Providing multiple wavelengths may be particularly beneficial whenexciting fluorescence in at least some of the particles in the coreflow, and detecting the fluorescence with the detector. Otherapplications may also benefit from a multiple wavelength light source.

FIG. 24 is similar to the illustrative embodiment shown in FIG. 23, butfurther shows a second optical element 730 between the movable opticalelement 704 and the flow stream 712. Optical element 730 may be adaptedto, for example, help columnate the light beam 702 before it engages thecore flow 706, regardless of the incident angle of the light beam 702.In some cases, this may help maintain a more consistent incident lightbeam on the core flow 706 regardless of the position of the core flow706 in the flow channel 712.

FIG. 25 is a schematic diagram showing yet another illustrativeembodiment of the present invention which uses a mechanical actuator toalign a light beam with the core flow of a flow stream. Thisillustrative embodiment includes a light source 750 for producing alight beam 752, a first optical element 754 for focusing the light beam752 on the core flow 756 of a flow stream, and a detector 758 fordetecting scattered light 760 from the core flow 756. In FIG. 25, asecond optical element 762 is provided between the core flow 756 and thedetector 758, but this is not required. The optical elements 754 and 762are shown schematically as lenses, but they may each include a singlelens, a set of lenses, or any other suitable optical element, asdesired.

As in FIGS. 23-24, the core flow 756 is included in a flow streamtraveling down a flow channel 764. The flow channel 764 shown in FIG. 25is flowing into the page. The core flow 756 may include a sheath fluid(liquid or gas) flowing on either side of the core flow 756. In someembodiments, the sheath fluid and core flow 756 have laminar flow asthey pass through the flow channel 764.

As shown generally at 770, the core flow 756 may be relatively centeredin the flow channel 764. However, under some conditions, the core flow756 may not flow down the center or at some other predetermined positionin the flow channel 764. For example, as shown generally at 772, thecore flow 756 may flow right of center of the flow channel 764.Likewise, although not shown, the core flow 756 may also flow left ofcenter of the flow channel 764.

To help compensate for the various possible positions of the core flow756 within the flow channel 764, it is contemplated that an actuator orthe like (not explicitly shown in FIG. 25) may be used to move theoptical element 754 and light source 750, generally shown at 774, sothat the light beam 752 emitted by the light source 750 is aligned with(e.g. focused on) the current position of the core flow 756 in the flowchannel 764. As in FIGS. 23-24, the actuator may be controlled by acontroller. In some cases, the controller may receive one or morefeedback signals indicating whether the light beam 752 is currentlyaligned with (e.g. focused on) the current position of the core flow 756in the flow channel 764. If not, and as shown generally at 772, thecontroller may instruct the actuator to move the optical element 754 andthe light source 750 until the light beam 752 is aligned with (e.g.focused on) the current position of the core flow 756 in the flowchannel 764.

Again, the actuator may be any type of mechanical actuator. In somecases, the actuator may be a stepper motor, a voice coil, anelectrostatic actuator, a magnetic actuator, a micro-positioningactuator similar to that shown and described in U.S. Pat. No. 6,445,514,or any other suitable actuator, as desired.

FIG. 26 is a schematic diagram showing another illustrative embodimentof the present invention which uses a mechanical actuator to align alight beam with the core flow of a flow stream. This illustrativeembodiment includes a light source 780 for producing a light beam 782, afirst optical element 784 for focusing the light beam 782 on the coreflow 786 of a flow stream, and a detector 788 for detecting scatteredlight 790 from the core flow 786. In FIG. 26, a second optical element792 is provided between the core flow 786 and the detector 788, but thisis not required. The optical elements 784 and 792 are shownschematically as lenses, but they may each include a single lens, a setof lenses, or any other suitable optical element, as desired.

The core flow 786 is included in a flow stream traveling down a flowchannel 794. In one illustrative embodiment, the flow channel 794 may bepart of, for example, a fluidic cartridge 800. The flow channel 794shown in FIG. 26 is flowing into the page. The core flow 786 may includea sheath fluid (liquid or gas) flowing on either side of the core flow786. In some embodiments, the sheath fluid and core flow 786 havelaminar flow as they pass through the flow channel 794.

As shown generally at 802, the core flow 786 may be relatively centeredin the flow channel 794. However, under some conditions, the core flow786 may not flow down the center or at some other predetermined positionin the flow channel 794. For example, as shown generally at 804, thecore flow 786 may flow left of center of the flow channel 794. Likewise,although not shown, the core flow 786 may also flow right of center ofthe flow channel 794.

To help compensate for the various possible positions of the core flow786 within the flow channel 794, it is contemplated that an actuator orthe like (not explicitly shown in FIG. 26) may be used to move the flowchannel 794, or in some cases the entire fluidic cartridge 800, so thatthe light beam 782 emitted by the light source 780 is aligned with (e.g.focused on) the current position of the core flow 786 in the flowchannel 794. As detailed above, the actuator may be controlled by acontroller. In some cases, the controller may receive one or morefeedback signals indicating whether the light beam 782 is currentlyaligned with (e.g. focused on) the current position of the core flow 786in the flow channel 794. If not, and as shown generally at 804, thecontroller may instruct the actuator to move the flow channel 794, or insome cases the entire fluidic cartridge 800, until the light beam 782 isaligned with (e.g. focused on) the current position of the core flow 786in the flow channel 794.

Again, the actuator may be any type of mechanical actuator. In somecases, the actuator may be a stepper motor, a voice coil, anelectrostatic actuator, a magnetic actuator, a micro-positioningactuator similar to that shown and described in U.S. Pat. No. 6,445,514,or any other suitable actuator, as desired.

FIG. 27 is a schematic diagram showing another illustrative embodimentof the present invention which uses a mechanical actuator to align alight beam with the core flow of a flow stream. This illustrativeembodiment includes a light source 900 for producing a light beam 902, afirst optical element 904 for focusing the light beam 902 on the coreflow (not explicitly shown in FIG. 27) in a flow channel 906, a secondoptical element 908 for focusing scattered light on a detector 910. Theillustrative embodiment shown in FIG. 27 is similar to that shown inFIG. 16. However, in some embodiments, the light source 902 in FIG. 27may include a single light source rather than an array of light sources.

The core flow is included in a flow stream traveling along a flowchannel 906. The flow channel 906 shown in FIG. 27 is flowing in anupward direction. The core flow may include a sheath fluid (liquid orgas) flowing on either side of the core. In some embodiments, the sheathfluid and core flow have laminar flow as they pass through the flowchannel 906.

As detailed above, the core flow may be relatively centered in the flowchannel 906. However, under some conditions, the core flow may not flowdown the center or at some other predetermined position in the flowchannel 906. For example, in the illustrative embodiment of FIG. 27, thecore flow may flow left of center or right of center of the flow channel906.

To help compensate for the various possible positions of the core flowwithin the flow channel 906, it is contemplated that an actuator or thelike (not explicitly shown in FIG. 27) may be used to move the opticalelement 904, as shown by dashed arrows 920 a and 920 b, so that thelight beam 902 emitted by the light source 900 is aligned with (e.g.focused on) the current position of the core flow in the flow channel906. The actuator may be controlled by a controller. In some cases, thecontroller may receive one or more feedback signals indicating whetherthe light beam 902 is currently aligned with (e.g. focused on) thecurrent position of the core flow in the flow channel 906. If not, thecontroller may instruct the actuator to move the optical element 904until the light beam 902 is aligned with (e.g. focused on) the currentposition of the core flow in the flow channel 906.

Like above, the actuator may be any type of mechanical actuator. In somecases, the actuator may be a stepper motor, a voice coil, anelectrostatic actuator, a magnetic actuator, a micro-positioningactuator similar to that shown and described in U.S. Pat. No. 6,445,514,or any other suitable actuator, as desired.

Having thus described the preferred embodiments of the presentinvention, those of skill in the art will readily appreciate that theteachings found herein may be applied to yet other embodiments withinthe scope of the claims hereto attached.

1. A detection system comprising: a light source arrangement forproviding a light beam proximate to a flow channel for containing a corestream; a detection mechanism proximate to the flow channel; and analignment affecter for shifting the light beam and a core streamrelative to each other; and wherein the detection mechanism may detectlight about alignment between the light beam and the core stream, anddetect light containing information about the core stream.
 2. The systemof claim 1, wherein the detection mechanism may detect light from thecore stream and convert the detected light into a first signal.
 3. Thesystem of claim 2, wherein the alignment affecter may align the lightbeam and the core stream according to the first signal.
 4. The system ofclaim 3, wherein information about the core stream may be providedaccording to the first signal.
 5. The system of claim 4, wherein: thecore stream comprises particles; and the information about the corestream may be about size, velocity, type, shape, structure, granularity,surface, antigens and the like of the particles.
 6. The system of claim4, wherein the light source arrangement comprises a plurality of lightsources.
 7. The system of claim 6, wherein a light source among theplurality of light sources may be selected for alignment of the lightbeam and the core stream.
 8. The system of claim 7, wherein theplurality of light sources comprises VCSELs.
 9. The system of claim 7,wherein the plurality of light sources comprises edge emitting lasers.10. The system of claim 7, wherein the plurality of light sourcescomprises LEDs.
 11. The system of claim 4, further comprising an opticalmechanism proximate to the light source arrangement.
 12. The system ofclaim 11, wherein the optical mechanism may be adjusted for alignment ofthe light beam and the core stream.
 13. The system of claim 4, whereinthe light source arrangement may be adjusted for alignment of the lightand the core stream.
 14. The method of claim 1, wherein the flow channelis of an instrument used for classifying blood cells.
 15. The method ofclaim 1, wherein the flow channel is of an instrument used foridentifying biological warfare agents.
 16. The method of claim 1,wherein the flow channel is of an instrument used for hematologyapplications.
 17. The method of claim 1, wherein the flow channel is ofan instrument used for identifying environmental particles.
 18. Themethod of claim 17, wherein the environmental particles may come fromair, water, food, soil, and the like.
 19. A method of alignment andparameter detection, comprising: emanating a light beam to a flowchannel; detecting light from a core stream in the flow channel;adjusting an alignment of the light beam and the core stream accordingto detected light; and obtaining information about the core stream fromdetected light.
 20. The method of claim 19, wherein the light foradjusting an alignment of the light beam and the core stream, and thelight for obtaining information about the core stream are detected byone detection mechanism.
 21. The method of claim 20, wherein thealignment is adjusted according to a magnitude of the detected light.22. The method of claim 21, wherein the flow channel is of an instrumentused for classifying blood cells.
 23. The method of claim 21, whereinthe flow channel is of an instrument used for identifying biologicalwarfare agents.
 24. The method of claim 21, wherein the flow channel isof an instrument used for hematology applications.
 25. The method ofclaim 21, wherein the flow channel is of an instrument used foridentifying environmental particles.
 26. The method of claim 25, whereinthe environmental particles may come from air, water, food, soil, andthe like.
 27. The method of claim 21, wherein the flow channel is of acytometer.
 28. A detection system comprising: a light source mechanismfor providing a light beam proximate to a flow channel; a detectionmechanism proximate to the flow channel; and an alignment mechanism foradjusting an alignment of the light beam and a core stream of the flowchannel; and wherein the detection mechanism may detect light indicativeof the alignment of the light beam and the core stream, and detect lightabout parameters of a core stream in the flow channel.
 29. The system ofclaim 28, further comprising a processor connected to the detectionmechanism and the alignment mechanism.
 30. The system of claim 29,wherein: the detection mechanism may convert detected light into a firstsignal; the processor may convert the first signal into a second signalindicative of an amount of change of position between the light beam andthe core stream to effect an alignment of the light beam and the corestream; the alignment mechanism may provide, according to the secondsignal, the amount of change of position between the light beam and thecore stream to effect an alignment of the light beam and the corestream; and the processor may convert the first signal into a thirdsignal indicative of parameters of the core stream.
 31. The system ofclaim 30, wherein the parameters may comprise size, velocity, type,shape, structure, granularity, surface, antigen, and the like aboutparticles of the core stream.
 32. Means for alignment and parameterdetection, comprising: means for emanating a light beam to a flowchannel; means for detecting light scattered by a core stream in theflow channel; means for determining an amount of alignment between thelight beam and the core stream according to detected light from themeans for detecting light; means for changing the amount of alignmentbetween the light beam and the core stream according to the detectedlight; and means for obtaining parameter information about the corestream from the detected light.
 33. The means of claim 32, wherein theparameter information may comprise size, velocity, type, shape,structure, granularity, surface, antigen, and the like about particlesof the core stream.
 34. The means of claim 33, wherein the flow channelis of an instrument used for classifying blood cells.
 35. The means ofclaim 33, wherein the flow channel is of an instrument used foridentifying biological warfare agents.
 36. The means of claim 33,wherein the flow channel is of an instrument used for hematologyapplications.
 37. The means of claim 33, wherein the flow channel is ofan instrument used for identifying environmental particles.
 38. Themeans of claim 37, wherein the environmental particles may come fromair, water, food, soil, and the like.
 39. The means of claim 33, whereinthe flow channel is of a cytometer.
 40. A detection system comprising: alight source mechanism for providing a light beam proximate to a flowchannel; a detection mechanism proximate to the flow channel; a firstalignment mechanism for adjusting an alignment of the light beamrelative to the flow channel, according to detected light from thedetection mechanism; a second alignment mechanism for adjusting analignment of the light beam relative to a core stream of the flowchannel, according to detected light from the detection mechanism; and aparameter mechanism for determining parameters of the core streamaccording to detected light from the detection mechanism.